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. 2010 Aug 4;99(3):887–896. doi: 10.1016/j.bpj.2010.04.076

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© 2010 by the Biophysical Society.

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Mutational epistasis determined from a comparison of thermal denaturation temperatures for each adaptive mutant with and without the AK_BSUBQ199R background. Circular dichroism was used to monitor unfolding as a function of temperature with data fitted as an apparent two-state transition. (a) Double mutants: AK_BSUB (open black circles), AK_BSUBQ199R (solid purple squares), AK_BSUBQ16L(open red squares), AK_BSUBT179I (yellow crosses), AK_BSUBA193V (solid green circles), AK_BSUBG213E (blue triangles), and AK_BSUBG214R (orange stars). (b) AK_BSUB (black), AK_BSUBQ199R (purple) compared to single mutants: AK_BSUB (open black circles), AK_BSUBQ199R/Q16L (open red squares); AK_BSUBQ199R/T179I (yellow crosses); AK_BSUBQ199R/A193V (solid green circles); and AK_BSUBQ199R/G214R (orange stars). (c) Point mutation at positions 213 and 214: AK_BSUB (open black circles), AK_BSUBQ199R (solid purple squares), AK_BSUBQ199R/G213A (teal crosses), AK_BSUBQ199R/G213Q (brown stars), AK_BSUBQ199R/G214A (solid gray circles), and AK_BSUBQ199R/G214K (open pink squares). (d) Far-UV wavelength scan of AK_BSUBQ199R (purple squares) and AK_BSUBG213E (blue triangles) at 20°C.